The human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (hA3G) belongs to a family of apolipoprotein B-editing catalytic polypeptide (APOBEC) proteins whose cytidine deaminase activity are cellular factors found to play a role in innate immunity.1,2 Other known members of this family include APOBEC1, 2, APOBEC3A-3H, and AID.3 hA3G has been found to induce dC-to-dU mutations in minus-strand DNA formed during reverse transcription, resulting in hypermutation of plus strand DNA. This enzymatic editing of HIV reverse transcripts leads to degradation of deaminated minus-strand DNA, thus restricting HIV type 1 (HIV-1) replication.2
HIV-1 and other lentiviruses encode the virion infectivity factor (Vif), an accessory protein that is critical for in vivo replication of HIV-1.4,5 Vif counteracts the antiviral activity of hA3G by preventing the virion incorporation of endogenous APOBEC3G, thereby effectively depleting the intracellular levels of this enzyme in HIV-1-infected T cells. The translation of APOBEC3G mRNA becomes impaired and its degradation by a ubiquitine-proteasome pathway is induced.6-8 However, Vif expression reduces hA3G levels in a dose-dependent manner: at lower Vif to hA3G ratios, it has no detectable effect on hA3G levels, whereas higher expression levels of hA3G can overcome the Vif-induced degradation.8
hA3G, hA3F, and hA3B are expressed in human cells, tissues, and peripheral blood mononuclear cells (PBMCs).9 As with hA3G, hA3F also induces G-to-A hypermutation, though both the dinucleotide target for deaminase and degree of mutagenesis are different between these 2 proteins in vitro observations.10 Another APOBEC protein, hA3B, also inhibits HIV-1 in cultured cells assayed. Other than hA3G, hA3B has, at most, a modest effect on HIV-1 infectivity. Furthermore, hA3B also differs from hA3F and hA3G in that it is unable to bind to HIV-1 Vif in coexpressing cells and is therefore efficiently packaged into HIV-1 virions regardless of Vif expression.11 It is therefore resistant to HIV Vif and is able to suppress the infectivity of both Vif-deficient and wild-type HIV-1 virus with equal efficiency. These studies on hA3G, hA3F, and hA3B underscore the need for further investigation of the significance in vivo infection, especially among individual differences in HIV disease progression.
The natural history of HIV-1 infection and progression to AIDS shows high variability. Slow progressors (SP) make up about 20% of HIV-infected individuals and are defined as those who remain free from AIDS-related diseases and maintain normal CD4+ T-cell counts for more than 10 years after infection without any antiretroviral therapy.12-14 Mechanisms for this slow progression are not well understood, however, the effects of hA3G, hA3F, and hA3B which block retroviral infection in vitro, may play a role. Recent studies investigating the relationship between mRNA level of hA3G and viral load and CD4+ T-cell counts have not delivered consistent results.15,16 The contribution of these cellular defense factors to HIV disease progression deserves more in vivo investigation, particularly in the case of hA3B which has not been examined to date.
This study uses real-time polymerase chain reaction (PCR) assay to quantify hA3G, hA3F, and hA3B mRNA levels in PBMCs of Chinese HIV-1-infected individuals and healthy HIV-negative controls. We hypothesize that due to the similarity of viral factors, the individual variation of hA3G, hA3F, and hA3B mRNA expression levels in different disease progression stages would represent, to some extent, the contribution of each cytidine deaminase to the disease progression. To test this, we determined the mRNA expression levels of hA3G, hA3F, and hA3B and measured the correlations between these mRNA levels, CD4+ T-cell counts, and HIV-1 viral loads to evaluate their respective roles in individual variations of disease progression rates.
PBMC samples of 46 HIV-infected and treatment-naive subjects and 17 HIV-uninfected healthy controls were recruited from Henan province after obtaining written informed consent. All subjects had been infected with B' subtype of HIV-1 via blood donation and were diagnosed as HIV-1 positive by confirmatory Western blot test. HIV-infected subjects were classified into 3 groups: slow progressors (SP), asymptomatic HIV-infected (AS), and AIDS patients (AIDS). Those who were infected with HIV-1 for more than 10 years with CD4+ T cells ≥500 cells per microliter and showed no HIV symptoms were classified as SP; those infected for more than 5 years with CD4+ T cells between 200 cells per microliter and 500 cells per microliter and without defined symptoms were classified as AS; and those with CD4+ T-cell counts <200 cells per microliter were classified as the AIDS group. All 17 healthy controls were HIV-antibody negative. HIV-1 B′ subtype was determined and confirmed by phylogenetic analysis based on segments of env, pol, and gag gene regions.
Determination of CD4+ T-Cell Counts
CD4+ T-cell count was measured using TriTEST CD4FITC/CD8PE/CD3PerCP reagent with 20 μL anticoagulated whole blood. After incubation for 15 minutes in the dark at room temperature, Fluorescence-activated cell sorting lysis solution was added, and the sample was then incubated for another 15 minutes in the dark at room temperature. CD4+ T-cell count and corresponding ratios were obtained by flow cytometer analysis with Fluorescence-activated cell sorting MULTISET software.
HIV Viral Load Assay
HIV RNA was extracted from plasma samples stored at −70°C and amplified by a standardized reverse transcription-PCR assay according to the manufacturer's instructions (COBAS Amplicor; HIV-1 Monitor Test Version 1.5; Roche Diagnostics, Branchburg, NJ). All viral load values were transformed to log10 values for statistical analysis.
Quantitative Real-time PCR
Whole blood was collected by venipuncture in vacutainer tubes containing EDTA (Becton Dickinson, Plymouth, UK), and PBMCs were separated with lymphocyte separation medium. RNA was isolated from 5 × 106 cells and treated with DNase (Qiagen, Hilden, Germany). cDNA products were synthesized with Random Primers and ImProm-II Reverse Transcriptase (Promega, Madison, WI). hA3G (NM_021822), hA3F (NM_145298), and hA3B (NM_004900) mRNA expression levels were quantified using TaqMan chemistry with a primer/probe combination to distinguish among highly homologous sequences of cytidine deaminases.
First-strand cDNA products were used in a 25 μL reaction mixture containing 2 × TaqMan Universal PCR Master Mix 12.5 μL and 20 × TaqMan Gene Expression Assay 1.25 μL. A commercially available primer/probe combination was used to quantify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a normalizing control sequence for the number of cell equivalents in total RNA starting material.
All reactions were run in an ABI 7500 analyzer, with 1 cycle at 50°C (2 minutes) followed by 95°C (10 minutes), and 40 cycles of 95°C (15 seconds) proceeded by 60°C (1 minute). Data were collected and analyzed using Sequence Detection software (Applied Biosystems, Foster City, CA). Absolute mRNA copy numbers were calculated by generating standard curves using serial dilutions of plasmids containing the desired gene (hA3G, hA3F, hA3B, or GAPDH ). Each sample was run in duplicate. hA3G, hA3F, hA3B mRNA expression levels were calculated as number of copies per 100,000 copies of GAPDH.
Statistical analyses were performed using SPSS 13.0. Differences between the groups were assessed using either a 2-sample t test for 2 groups or a 1-way Analysis of Variance and subsequent Student-Newman-Keuls test for more than 2 groups. The viral loads data were transformed into log10 before analysis, and the mRNA copies of hA3G, A3B, and A3F were calculated as copies per 100,000 copies of GAPDH mRNA and then log transformed (base e) for statistical analysis. The means and standard deviations were used for comparison. Correlation between 2 quantitative variances was determined using the Pearson correlation coefficient. A P value of <0.05 was considered statistically significant.
Forty-six HIV-infected patients consisting of 18 SP, 19 AS; 9 AIDS; and 17 HIV-uninfected healthy controls were investigated in our study (Table 1). Analysis of hA3G, hA3B, and hA3F levels revealed significant differences between HIV(+) and HIV(−) subjects in terms of hA3G (t = −4.887, P < 0.001) and hA3B (t = −2.168, P = 0.045) levels but not for hA3F (t = −1.666, P = 0.109). The results showed that expression levels of hA3G were higher in SP than in AS or AIDS (P < 0.05 ), that hA3B levels were higher in SP than in AIDS (P < 0.05), and that hA3B levels in AS were also higher than in AIDS (P < 0.05 ). Regarding different mRNA levels among the APOBEC proteins in HIV-infected subjects, hA3B expression levels were found to be significantly lower than those of hA3F and hA3G (P < 0.001), whereas there was no statistically significant difference between the levels of expression of hA3F and hA3G (Table 2).
A comparison of coexpression of these proteins in HIV-infected patients and healthy controls revealed a strong positive correlation among these 3 gene mRNA expressions (Fig. 1).
CD4+ Cell Counts and Expression Levels of hA3G, hA3B, and hA3F mRNA
Pearson correlation tests revealed that hA3G and hA3B expression levels were both positively correlated with CD4+ T-cell counts (r = 0.436, P = 0.002 and r = 0.334, P = 0.025, respectively) and that there was no correlation between hA3F mRNA level and CD4+ cell counts (r = 0.104, P = 0.490) (Fig. 2).
Viral Load and Expression Levels of hA3G, hA3B, and hA3F mRNA
The results showed that hA3G and hA3B mRNA expression levels were negatively correlated with viral loads (r = −0.306, P = 0.038, r = −0.301, P = 0.044) respectively. There was no statistical correlation between hA3F mRNA levels with viral loads (r = −0.265, P = 0.075) (Fig. 3).
We examined potential role of hA3G, hA3F, and hA3B in HIV disease progression by measuring the mRNA levels of these proteins and their correlation with CD4+ T-cell counts and HIV-1 viral loads. Our results suggest a link between APOBEC protein expression and disease progression: hA3G and hA3B levels were consistently lower in HIV-positive patients as compared with HIV-negative healthy controls, and hA3G and hA3B levels were significantly higher in SP compared with AIDS patients; hA3G and hA3B levels were positively correlated with CD4+ T-cell counts but negatively correlated with viral loads in HIV/AIDS patients. However, Ulenga et al17 found that the expression of hA3G and hA3F increase after infection. This possibly due to the increase of interferon-α upon HIV-1 infection which has been found to elevate levels of hA3G expression in the early stages of disease progression.18,19 On the other hand, the infection period in subjects of both ours and Cho et al's are longer, particularly in our study, which are all more than 7 years. This may explain the difference between the results of ours and Ulenga's. As the disease progresses, however, there is greater variance in hA3G levels among HIV-1-infected individuals which is shown in our study. However, the differences in hA3G mRNA expression between HIV-infected and HIV-negative healthy controls remain to be explained by further studies.
Jin et al16 found a significant inverse correlation between hA3G mRNA levels with HIV viral loads and a highly significant positive correlation with CD4+ T-cell count in highly active antiretroviral therapy (HAART)-naive subjects. Adversely, Cho et al15 did not find correlation between hA3F or hA3G mRNA levels and viral loads or CD4+ T-cell counts in HIV-positive subjects. The opposite conclusion might come from sample treatment or subjects with or without HAART therapy. In study of Jin et al, CD4+ T cells were costimulated with anti-CD3 and anti-CD28 antibodies before RNA extraction.16 In study of Cho,15 HIV-infected individuals had not taken HAART therapy for at least 3 months. HAART therapy may improve the cell immunity status of patients, such as increasing the production of cytokines. Studies have shown that the expression of hA3G mRNA could be induced by cytokines.20 Considering possible effects of HAART and antibody stimulation on the expression of cytidine deaminases, we selected HAART-naive individuals and extracted RNA without any treatment on PBMCs. The APOBEC proteins mRNA expression levels investigated represent the natural physiologic steady state in vivo and their true significance for disease progression.
Based on the expression differences of hA3G among SP, AS, and AIDS groups and the correlation of it with CD4+ T-cell counts, and HIV viral loads, the results have provided evidence that the expression levels of hA3G mRNA are negatively associated with HIV disease progression, and higher levels of hA3G mRNA may play an important role in controlling HIV infection in vivo. When hA3G is expressed in high levels, it could overcome the degradation of HIV-1 Vif and inhibit HIV-1 replication in vivo. Thus, with fewer viruses released, less CD4+ cells are damaged, which may in turn delay the progression rate, as observed in the SP group. However, more in vitro and in vivo studies on the intrinsic mechanisms need to be performed.
hA3B has similar aminoacid sequences with hA3G proteins. A report found that hA3B mRNA was not detectable in primary tissues that coexpress hA3G and hA3F, and hA3B mRNA was also not detected in a range of primary human tissue samples including PBMCs when using semiquantitative reverse transcription-PCR assay. However, when we used real-time quantitative PCR to quantitative hA3B mRNA, we found that it was expressed in PBMCs but at lower levels compared with hA3G and hA3F. This result is encouraging because although hA3B has, at most, a modest effect on HIV-1 infectivity, it inhibits both Vif-deficient and wild-type HIV-1 virus in vitro. Furthermore, this inhibition is not influenced by Vif expression.11 The results in our study showed that the hA3B mRNA levels of SP are significantly higher than that of AIDS subjects. The hA3B mRNA levels in HIV/AIDS patients are positively correlated with CD4+ T-cell counts and negatively with viral loads. Our results suggest that hA3B is also associated with HIV disease progression and could be another protective factor.
There was no statistical difference in hA3F mRNA levels among these groups, and there was no correlation between hA3F mRNA levels with CD4+ T-cell count or viral load. These results suggest that hA3F is most likely not associated with HIV disease progression. Studies have shown that hA3F is a strong inhibitor of Vif-deficient HIV-1, though not as potent as A3G. A3F is also efficiently inhibited by HIV-1 Vif via proteasomal degradation. However, the domains interacted with HIV-1 Vif, and the dinucleotide target for deaminase, and degree of mutagenesis are different between hA3G and hA3F.21-22 Consequently, their significance in vivo are not the same as our results have provided.
To investigate if these 3 genes are coexpressed in vivo, the correlation between their expression levels were analyzed. We found that there were significantly positive correlations with one another. However, as mentioned, the expression levels of hA3B mRNA is lower than that of hA3G and hA3F.
There has been an increasing focus on hA3G and its family members due to their innate antiretroviral functions. Our results illustrate that when the hA3G and hA3B mRNA expression levels of HIV/AIDS patients are higher, their CD4+ T-cell counts are higher and their viral loads are lower. As such, modulating the mRNA expression of hA3G and hA3B and increasing the syntheses of their respective active proteins may be an effective way to control viremia and the replication of HIV in CD4+ T cells in vivo. Because our results focused on the mRNA level, the protein levels of hA3G and hA3B in CD4+ T cells of HIV-infected individuals and their enzyme activities are yet unknown, and this area deserves further study.
The authors thank Kumi Smith and Naomi Juniper for their editing assistance.
1. Sheehy AM, Gaddis NC, Choi JD, et al. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral vif protein. Nature
2. Harris RS, Bishop KN, Sheehy AM, et al. DNA deamination mediates innate immunity to retroviral infection. Cell
3. Cullen BR. Role and mechanism of action of the APOBEC3 family of antiretroviral resistance factors. J Virol
4. Fisher AG, Ensoli B, Ivanoff L, et al. The sor gene of HIV-1 is required for efficient virus transmission in vitro. Science
5. Strebel K, Daugherty D, Clouse K, et al. The HIV ‘A’ (sor) gene product is essential for virus infectivity. Nature
6. Yu X, Yu Y, Liu B, et al. Induction of APOBEC3G ubiquitination and degradation by an HIV-1 Vif-Cul5-SCF complex. Science
7. Bishop KN, Holmes RK, Sheehy AM, et al. Cytidine deamination of retroviral DNA by diverse APOBEC proteins. Curr Biol
8. Mehle A, Strack B, Ancuta P, et al. Vif overcomes the innate antiviral activity of APOBEC3G by promoting its degradation in the ubiquitin-proteasome pathway. J Biol Chem
9. Jarmuz A, Chester A, Bayliss J, et al. An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22. Genomics
10. Wiegand HL, Doehle BP, Bogerd HP, et al. A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins. EMBO J
11. Doehle BP, Schäfer A, Cullen BR. Human APOBEC3B is a potent inhibitor of HIV-1 infectivity and is resistant to HIV-1 Vif. Virology
12. Hogan CM, Hammet SM. Host determinants in HIV infection and disease Part 1: cellular and humoral immune response. Ann Intern Med
13. Bakari M, Urassa W, Mhalu F, et al. Slow progression of HIV-1 infection in a cohort of antiretroviral naïve hotel workers in Dar es Salaam, Tanzania as defined by their CD4 cell slopes. Scand J Infect Dis
14. Langford SE, Ananworanich J, Cooper DA, et al. Predictors of disease progression in HIV infection: a review. AIDS Res Ther
15. Cho SJ, Drechsler H, Burke RC, et al. APOBEC3F and APOBEC3G mRNA levels do not correlate with human immunodeficiency virus type 1 plasma viremia or CD4+T-cell count. J Virol
16. Jin X, Brooks A, Chen H, et al. APOBEC3G/CEM mRNA (hA3G)levels associate inversely with human immunodeficiency virus viremia. J Virol
17. Ulenga NK, Sarr AD, Thakore-Meloni S, et al. Relationship between human immunodeficiency type 1 infection and expression of human APOBEC3G and APOBEC3F. J Infect Dis
18. Baca-Regen L, Heinzinger N, Stevenson M, et al. Alpha interferon-induced antiretroviral activities: restriction of viral nucleic acid synthesis and progeny virion production in human immunodeficiency virus type 1-infected monocytes. J Virol
19. Chen K, Huang J, Zhang C, et al. Alpha interferon potently enhances the anti-human immunodeficiency virus type 1 activity of APOBEC3G in resting primary CD4 T cells. J Virol
20. Stopak KS, Chiu YL, Kropp J, et al. Distinct patterns of cytokine regulation of APOBEC3G expression and activity in primary lymphocytes, macrophages, and dendritic cells. J Biol Chem
21. Russell RA, Smith J, Barr R, et al. Distinct domains within APOBEC3G and APOBEC3F interact with separate regions of HIV-1 Vif. J Virol
22. Zennou V, Bieniasz PD. Comparative analysis of the antiretroviral activity of APOBEC3G and APOBEC3F from primates. Virology
Keywords:© 2010 Lippincott Williams & Wilkins, Inc.
hA3G; hA3B; hA3F; HIV-1; slow progress patients